U.S. patent application number 10/608536 was filed with the patent office on 2004-03-11 for antibodies specific to human prostacyclin synthase.
Invention is credited to Tanabe, Tadashi.
Application Number | 20040049015 10/608536 |
Document ID | / |
Family ID | 14634811 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040049015 |
Kind Code |
A1 |
Tanabe, Tadashi |
March 11, 2004 |
Antibodies specific to human prostacyclin synthase
Abstract
A DNA-comprising a DNA having a nucleotide sequence encoding the
amino acid sequence of human-originated prostacyclin synthase
(PGIS) substantially depicted in Sequence No. 12, a vector
comprising said DNA, a host cell transformed with said vector and a
method for preparing human-originated PGIS comprising culturing
said host cell in a medium. A polypeptide having the amino acid
sequence of human-originated PGIS substantially depicted in
Sequence No. 12 and an antibody having a reactivity with said
human-originated PGIS. A pharmaceutical composition comprising said
DNA or a vector comprising said DNA. A method for promoting the
production of PGI.sub.2 and a method for treating the diseases
induced by a low production of PGI.sub.2, comprising introducing
said DNA or a vector comprising said DNA into human or other
animals. The present invention clarifies the primary structure of
human-originated PGIS and the nucleotide sequence encoding same.
The PGIS and its DNA are useful as reagents for the development of
therapeutic agents for the cardiovascular diseases induced by the
production imbalance between PGI.sub.2 and TXA.sub.2, and as
diagnostics for determining the in vivo tissue expression level-and
distribution of PGIS or mRNA thereof. Moreover, they can be used as
therapeutic agents for cardiovascular diseases, which introduce
PGIS and the like into human or other animals in a lesion-specific
manner. The production method of the present invention is useful
for the easy and efficient mass production of the human-originated
PGIS. The antibody of the present invention is useful for the
purification of the human-originated PGIS and immunohistochemical
analysis of the cause of a disease.
Inventors: |
Tanabe, Tadashi; (Osaka,
JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20037
US
|
Family ID: |
14634811 |
Appl. No.: |
10/608536 |
Filed: |
June 30, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10608536 |
Jun 30, 2003 |
|
|
|
09670582 |
Sep 27, 2000 |
|
|
|
09670582 |
Sep 27, 2000 |
|
|
|
09037758 |
Jan 10, 1998 |
|
|
|
09037758 |
Jan 10, 1998 |
|
|
|
08578709 |
Dec 28, 1995 |
|
|
|
5814509 |
|
|
|
|
Current U.S.
Class: |
530/388.26 ;
435/193; 435/320.1; 435/325; 435/6.14; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 9/00 20180101; C12N 9/90 20130101; A61K 48/00 20130101 |
Class at
Publication: |
530/388.26 ;
514/044; 435/006; 435/069.1; 435/193; 435/320.1; 435/325;
536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00; C07K 016/40; C12N 009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 1994 |
JP |
114316/1994 |
Claims
What is claimed is
1. A DNA comprising a DNA having a nucleotide sequence encoding an
amino acid sequence of a human-originated prostacyclin synthase
substantially depicted in Sequence Listing, Sequence No. 12.
2. The DNA of claim 1, comprising a DNA having a 28th-1527th
nucleotide sequence substantially shown in Sequence Listing,
Sequence No. 11.
3. The DNA of claim 2, comprising a DNA having a 28th-1527th
nucleotide sequence shown in Sequence Listing, Sequence No. 11.
4. A polypeptide comprising an amino acid sequence of a
human-originated prostacyclin synthase substantially shown in
Sequence Listing, Sequence No. 12.
5. The polypeptide of claim 4, comprising an amino acid sequence of
a human-originated prostacyclin synthase shown in Sequence Listing,
Sequence No. 12.
6. A recombinant vector comprising the DNA of any one of claims 1
to 3.
7. A host cell transformed with the recombinant vector of claim
6.
8. A transformed cell identified by International Deposit No. FERM
BP-4653 or FERM BP-4654.
9. A method for preparing a human-originated prostacyclin synthase,
comprising culturing the host cell of claim 7 in a medium and
recovering a human-originated prostacyclin synthase from the
obtained culture.
10. An antibody having a reactivity with a human-originated
prostacyclin synthase comprising an amino acid sequence
substantially shown in Sequence Listing, Sequence No. 12.
11. A pharmaceutical composition comprising the DNA of any one of
claims 1 to 3 and a pharmaceutically acceptable carrier.
12. A pharmaceutical composition comprising the recombinant vector
of claim 6 and a pharmaceutically acceptable carrier.
13. A pharmaceutical composition for promoting prostaglandin
I.sub.2 production, comprising the DNA of any one of claims 1 to 3
and a pharmaceutically acceptable carrier.
14. A pharmaceutical composition for promoting prostaglandin
I.sub.2 production, comprising the recombinant vector of claim 6
and a pharmaceutically acceptable carrier.
15. A pharmaceutical composition for treating a disease induced by
a low production of prostaglandin I.sub.2, comprising the DNA of
any one of claims 1 to 3 and a pharmaceutically acceptable
carrier.
16. A pharmaceutical composition for treating a disease induced by
a low production of prostaglandin I.sub.2, comprising the
recombinant vector of claim 6 and a pharmaceutically acceptable
carrier.
17. A method for promoting prostaglandin I.sub.2 production,
comprising introducing the DNA of any one of claims 1 to 3 into a
human or an animal.
18. A method for promoting prostaglandin I.sub.2 production,
comprising introducing the recombinant vector of claim 6 into a
human or an animal.
19. A method for treating a disease induced by a low production of
prostaglandin I.sub.2, comprising introducing the DNA of any one of
claims 1 to 3 into a human or an animal.
20. A method for treating a disease induced by a low production of
prostaglandin I.sub.2, comprising introducing the recombinant
vector of claim 6 into a human or an animal.
Description
TECHNICAL FIELD
[0001] The present invention relates to a polypeptide having an
amino acid sequence of human-originated prostacyclin synthase
(hereinafter referred to as PGIS), a DNA encoding same, a vector
containing said DNA, a host cell transformed with said vector and a
method for preparing human-originated PGIS comprising culturing
said host cell. The present invention also relates to an antibody
having a reactivity with said PGIS or its fragment. Moreover, the
present invention relates to a pharmaceutical composition
comprising said DNA or a vector containing said DNA, a method for
promoting the production of prostaglandin I.sub.2 and a method for
the treatment of the diseases induced by a low production of
prostaglandin I.sub.2.
BACKGROUND ART
[0002] PGIS is mainly contained in microsomal fractions of vascular
endothelial cells, and is an enzyme that catalyzes synthesis of
prostaglandin I.sub.2 (hereinafter referred to as PGI.sub.2), that
is, conversion of prostaglandin H.sub.2 (hereinafter referred to as
PGH.sub.2) to PGI.sub.2.
[0003] PGI.sub.2 synthesized by this enzyme has potent platelet
aggregation-inhibitory action and vascular smooth muscle-relaxing
action. On the other hand, platelets contain thromboxane A.sub.2
(hereinafter referred to as TXA.sub.2) having strong platelet
aggregation action and vascular smooth muscle-contracting action,
and the both substances act antagonistically in the vascular system
to maintain homeostasis [British Journal of Pharmacology,.vol. 76,
p 3 (1982)].
[0004] Cardiovascular diseases such as myocardial infarction,
thrombosis and arteriosclerosis, which are among the adult
diseases, have recently been considered to be caused by the
imbalance in the vascular production of PGI.sub.2 and TXA.sub.2,
particularly, insufficient vascular function due to low production
of PGI.sub.2 (ibid.).
[0005] For the therapeutic treatment of the diseases presumably
induced by the low production of PGI.sub.2, PGI.sub.2 may be
supplemented as a pharmaceutical product from the outside of the
body. However, PGI.sub.2 is chemically extremely unstable to the
extent that a practical use of PGI.sub.2 itself as a pharmaceutical
product may be unrealizable. In view of such situation, for
example, stable PGI.sub.2 analogs such as blood coagulation
inhibitor or vasodilator are under development.
[0006] The homeostasis in human and other animals which is
inherently based on the balance between PGI.sub.2 and TXA.sub.2 may
possibly destroyed by the administration of stable PGI.sub.2
analogs. That is, administration of stable PGI.sub.2 analogs in
large amounts is associated with a risk of lowering the
responsiveness of cells to PGI.sub.2, thus impairing its capability
of responding to PGI.sub.2 when such responsiveness is in urgent
need (Prostaglandins, vol. 19, p 2.(1980)].
[0007] For correcting the imbalance between PGI.sub.2. and
TXA.sub.2 and attempting the recovery of normal functions of the
vascular system in an expectation of therapeutic effect over
thrombosis and the like, chemically stable analogs may be used.
Alongside therewith, moreover, elucidation of physicochemical
property and biological property of PGIS, clarification of the
relations between PGIS production and PGI.sub.2 production while
using said PGIS or DNA encoding PGIS as a research sample, and
development of said PGIS or DNA encoding PGIS as pharmaceutical
products to regulate the production of PGI.sub.2 are considered to
be important and significant for the treatment of the above-said
various diseases caused by the imbalance between PGI.sub.2 and
TXA.sub.2.
[0008] Conventionally, there has been reported the tissue
distribution of PGIS, namely, its presence in -vascular endothelial
cells, non-vascular smooth muscle cells and arterial smooth muscle
of various organs [Advances in Prostaglandin, Thromboxane, and
Leukotriene Research, vol. 11, pp. 87-92 (1983) and J. Biol. Chem.,
vol. 258, No. 9, pp. 5922-5926 (1983)]. Meanwhile, isolation and
purification of PGIS from porcine and bovine have been tried
[porcine: Cytochrome P450, Biochemistry, Biophysics and
Environmental Implications, pp. 103-106 (1982); bovine: J. Biol.
Chem., vol. 258, No. 9, pp. 3285-3293 (1983)] and N-terminal amino
acid sequence and partial downstream amino acid sequence of bovine
PGIS have been reported [Advances in Prostaglandin, Thromboxane,
and Leukotriene Research, vol. 17, pp. 29-33 (1987) and Biochemical
and Biophysical Research Communications, vol. 197, No. 3, pp.
1041-1048 (1993)].
[0009] However, isolation, purification and amino acid sequence of
human PGIS have not been elucidated.
DISCLOSURE OF THE INVENTION
[0010] An object of the present invention is to clarify an amino
acid sequence of PGIS derived from human and provide said
human-originated PGIS and DNA encoding said PGIS.
[0011] Said PGIS and DNA encoding said PGIS are useful as reagents
for {circle over (1)} the analysis of the physicochemical and
biological properties of PGIS at the molecular or genetic level;
{circle over (2)} the analysis of the mechanism controlling the
production of PGIS and the mechanism controlling the production of
PGI.sub.2 by PGIS; and {circle over (3)} the investigation of the
cause of various cardiovascular diseases considered to be induced
by the production imbalance between PGI.sub.2 and TXA.sub.2, and
the molecular or genetic level analysis for the development of
therapeutic agents for said diseases. In addition, PGIS and its
mRNA are useful as diagnostics for the determination of expression
level and distribution in the body tissues. Still further, they are
expected to provide therapeutic agents for, for example, various
cardiovascular disorders such as thrombosis, myocardial infarction,
arteriosclerosis and angina pectoris, which enhance the production
level of PGI.sub.2 upon introduction of themselves, fragment
thereof or modified compound thereof into the body in a
lesion-specific manner.
[0012] Another object of the present invention is to provide a
recombinant vector containing a DNA encoding human-originated PGIS,
the expression system of PGIS which comprises a host cell
transformed with said vector, and a method for preparing PGIS by
genetic engineering using said expression system.
[0013] According to such method, human-originated PGIS can be
prepared in great amounts with ease and with high efficiency.
[0014] The present invention also aims at providing a
human-originated PGIS antibody useful for the purification of
human-originated PGIS and immunohistochemical analysis of the cause
of a disease.
[0015] The present inventor has conducted intensive studies with
the aim of accomplishing the above-mentioned objects, and succeeded
in cloning cDNA encoding PGIS from human aorta endothelial cells
and identifying the primary structure of human-originated PGIS from
the nucleotide sequence of said cDNA, which resulted in the
completion of the present invention.
[0016] Accordingly, the present invention relates to a DNA
comprising a DNA having a nucleotide sequence encoding an amino
acid sequence of human-originated PGIS substantially depicted in
Sequence No. 12, preferably a DNA comprising a DNA having a
28th-1527th nucleotide sequence substantially shown in Sequence No.
11, and more preferably a DNA having a 28th-1527th nucleotide
sequence shown in Sequence No. 11.
[0017] The present invention also-relates to a recombinant vector
comprising the above-mentioned DNA, a host cell transformed with
said vector and a method for preparing human-originated PGIS
comprising culturing said host cell in a medium and recovering
human-originated PGIS from the obtained culture.
[0018] The present invention also relates to a polypeptide having
an amino acid sequence of human-originated PGIS which is
substantially shown in Sequence No. 12, and antibodies having
reactivities with said human-originated PGIS.
[0019] The present invention further relates to a pharmaceutical
composition comprising said DNA or a recombinant vector comprising
said DNA. Said pharmaceutical composition can be used as a
medicament for promoting PGI.sub.2 production or for treating the
diseases induced by a low production of PGI.sub.2.
[0020] The present invention moreover relates to a method for
promoting the production of PGI.sub.2, comprising introducing the
above-mentioned DNA or a recombinant vector comprising said DNA
into human or other animals. The present invention also relates to
a method for treating the diseases induced by a low production of
PGI.sub.2, comprising introducing the above-mentioned DNA or a
recombinant vector comprising said DNA into human or other
animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows a restriction enzyme map of human PGIS cDNA,
and the PGIS DNA region comprised in .lambda. hPGIS141, pHPGIS135
and pHPGIS36.
[0022] FIG. 2 shows a restriction enzyme map of plasmid
pHPGIS36.
[0023] FIG. 3 shows a restriction enzyme map of plasmid
pHPGIS135.
[0024] FIG. 4 shows a restriction enzyme map of plasmid
pHPGIS1.
[0025] FIG. 5 shows a restriction enzyme map of human PGIS
expression vector pCMV-HPGIS1.
[0026] FIG. 6 shows an expression vector pUC-CAGGS.
[0027] FIG. 7 is a photograph showing the results of the analysis,
by thin layer chromatography, of the PGIS activity in the cells
into which pCMV-HPGIS1 has been introduced.
[0028] FIG. 8 is a photograph showing the results of the analysis,
by thin layer chromatography, of the PGIS activity in positive
control (bovine platelet microsomes).
[0029] FIG. 9 is a photograph showing the results of the analysis,
by thin layer chromatography, of negative control wherein pCMV
alone was introduced.
[0030] FIG. 10 is a graph showing the effects of the introduction
of human PGIS expression vector on the blood vessel smooth muscle
cell proliferation.
[0031] FIG. 11 is a photograph showing the results of RNA blot
(electrophoresis) analysis of human pGIS mRNA treated with
cytokines.
[0032] FIG. 12 is a photograph showing the distribution of PGIS
mRNA expression in human body (pancreas, kidney, skeletal muscle,
liver, lung, placenta, brain and heart) by electrophoresis.
[0033] FIG. 13 is a photograph showing the distribution of PGIS
mRNA expression in human body (peripheral leukocyte, large
intestine, small intestine, ovary, testicle, prostate, thymus and
spleen) by electrophoresis.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is explained in detail in the
following.
[0035] The polypeptide of the present invention has a catalytic
activity to convert PGH.sub.2 to PGI.sub.2 and has an amino acid
sequence of human-originated PGIS substantially shown in Sequence
Listing, Sequence No. 12 to be mentioned later.
[0036] By "substantially" is meant that the polypeptide of the
present invention is not limited to the polypeptide having the
amino acid sequence shown in Sequence No. 12, but may include
deletion, substitution and addition with respect to some of the
amino acids in the amino acid sequence shown in Sequence No. 12, as
long as the polypeptide has immunological and biological activity
(human PGIS activity) similar to that of human-originated PGIS
having said amino acid sequence.
[0037] While the site of deletion, substitution and addition of the
amino acids is not particularly limited, at least 441st Cys residue
and thereabout region in the amino acid sequence shown in Sequence
No. 12 need to be reserved. This is because human-originated PGIS
of the present invention is homologous to known cytochrome P450 in
the amino acid sequence, since it has Cys residue in the C-terminal
side of the amino acid sequence constituting the heme-binding site
(fifth ligand) which is important for the expression of biological
activity of cytochrome P450, and speculated to be a new protein
belonging to the cytochrome P450 family [see Seibutsu Butsuri, vol.
32, No. 1, pp. 10-15 (1992)].
[0038] The polypeptide of the present invention preferably has an
amino acid sequence of human-originated PGIS shown in Sequence No.
12.
[0039] The PGIS activity possessed by the polypeptide of the
present invention is a catalytic activity to convert PGH.sub.2 to
PGI.sub.2. Said PGIS activity can be determined according to the
method of Salmon, J. A. and Flower, R. J. et al [Methods Enzymol.,
86, pp. 91-99 (1982)] wherein the conversion of .sup.14C-labeled
PGH.sub.2 to PGI.sub.2 is assayed by separating the metabolite of
6-keto-PGF.sub.1.alpha. by thin layer chromatography and detecting
the radioactivity of said 6-keto-PGF.sub.1.alpha..
[0040] The present invention also relates to a DNA comprising a DNA
having a nucleotide sequence encoding the amino acid sequence of
human-originated. PGIS substantially shown in Sequence No. 12.
[0041] Said DNA may be any as long as it comprises a DNA having a
nucleotide sequence encoding the aforementioned amino acid sequence
of human-originated PGIS, and is exemplified by a DNA encoding the
polypeptide having the amino acid sequence shown in Sequence No. 12
or a polypeptide having the equivalent immunological and biological
activity. More specifically, it is a DNA comprising the 28th-1572nd
nucleotide sequence in the nucleotide sequence shown in Sequence
No. 11.
[0042] In general terms, the genetic recombinant technique enables
conversion of at least one nucleotide of a DNA sequence of a gene
to a different nucleotide according to the degeneracy of the
genetic code, without changing the amino acid sequence of a protein
produced by the gene. Accordingly, the DNA of the present invention
encompasses a DNA comprising a nucleotide sequence obtained by
modification for substitution, based on the genetic code, of the
28th-1527th nucleotide sequence of Sequence Listing Sequence No.
11.
[0043] The DNA of the present invention can be obtained by any
method. For example, the present invention encompasses
complementary DNA (cDNA) prepared from mRNA, DNA prepared from
genomic DNA, DNA obtained by chemical synthesis, DNA obtained by
amplification by PCR using RNA or DNA as a template, and DNA
constructed by suitably combining these methods.
[0044] The DNA of the present invention can be obtained by a method
comprising cloning cDNA from mRNA of human-originated PGIS by a
conventional method, a method comprising splicing an isolated
genomic DNA for PGIS, a method comprising chemical synthesis or
other method.
[0045] (1) For example, a method for cloning cDNA from mRNA
encoding human-originated PGIS comprises the following steps.
[0046] Cells producing (secreting) human-originated PGIS, such as
human aorta endothelial cells are cultured and mRNA encoding said
PGIS is prepared from the culture thereof. mRNA is prepared by, for
example, applying entire RNA prepared by a known method such as
guanidine thiocyanate method [Chirgwin, J. M. et al., Biochem., 18,
5294 (1979)], heat phenol method and AGPC to affinity
chromatography using oligo(dT)-cellulose or poly U-sepharose.
[0047] Using the obtained mRNA as a template, cDNA chain is
synthesized by a known method using a reverse transcriptase [e.g.,
the method of Okayama, H. et al: Okayama, H. et al., Mol. Cell.
Bio., 2, 161 (1982) and ibid. 3, 280 (1983)], and the method of
Gubler, U. and Hoffman, B. J.: Gubler, H. and Hoffman, B. J., Gene,
25. 263 (1983)], thereby converting the same to a double stranded
cDNA. This cDNA is inserted into a plasmid vector or a phage
vector, with which Escherichia coli is transformed, or transfected
after in vitro packaging, to prepare cDNA library.
[0048] The plasmid vector used here is not subject to any
particular limitation as long as it can be retained by replication
in the host, and the phage vector is not limited either as long as
it can proliferate in the host. Examples of the conventionally-used
cloning vector include pUC119, .lambda.gt10 and .lambda.gt11. When
immunological screening to be mentioned later is to be employed,
the vector preferably contains a promoter capable of expressing the
PGIS gene in the host.
[0049] The method for insertion of a cDNA into plasmid is
exemplified by a method described in Maniatis, T. et al [Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, p. 239
(1982)]. The method for insertion of a cDNA into phage vector
includes the method of Hyunh, T. V. et al [DNA Cloning, a practical
approach, 1, 49 (1985)]. For simplification, a commercially
available ligation kit (e.g., those manufactured by Takara Shuzo)
can be used. The recombinant plasmid and phage vector thus obtained
are introduced into a suitable host such as prokaryotic cells
(e.g., E. coli HB101, DH5 and MC1061/P3).
[0050] The method for introducing a plasmid into a host includes
calcium chloride method and calcium chloride/rubidium chloride
method described in Maniatis, T. et al [Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, p. 239 (1982)]
and electroporation method. The method for introducing a phage
vector into a host is exemplified by a method comprising in vitro
packaging of phage DNA and introducing same into proliferated host
cells. In vitro packaging can be carried out easily by using a
commercially available in vitro packaging kit (e.g., product of
Stratagene and product of Amersham).
[0051] The cDNA encoding the PGIS of the present invention can be
isolated from the cDNA library prepared by the above method, by a
combination of general cDNA screening methods.
[0052] Such methods include, for example, a method wherein an
oligonucleotide considered to be corresponding to the amino acid
sequence of human PGIS is chemically synthesized separately and
labeled with .sup.32P to give a probe, and a clone having the
desired cDNA is screened by a known colony hybridization
[Crunstein, M. and Hogness, D. S., Proc. Natl, Acid. Sci. USA, 72,
3961 (1975)] or plaque hybridization [Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, p. 239 (1982)];
and a method wherein PCR primer is prepared and a specific region
of PGIS is amplified by PCR method, which is followed by selecting
a clone having a DNA fragment encoding said region. When a cDNA
library prepared using a vector (e.g. .lambda.gt11 phage vector)
capable of expressing cDNA is used, the objective clone can be
selected based on an antigen-antibody reaction using the PGIS
antibody of the present invention to be mentioned later. When large
amounts of clone are treated, screening based on PCR is
preferable.
[0053] The nucleotide sequence of DNA thus obtained can be
determined by Maxam-Gilbert method [Maxam, A. M. and Gilbert, W.,
Proc. Natl. Acad. Sci. USA., 74, 560 (1977)] or synthetic
dideoxynucleotide chain termination method using phage M13 [Sanger,
F. et al, Proc. Natl. Acad. Sci. USA., 74, 5463-5467 (1977)]. The
PGIS gene can be obtained by cleaving all or part thereof from the
clone obtained above by using a restriction enzyme and the
like.
[0054] (2) A preparation method comprising isolating DNA encoding
PGIS from genomic DNA of human aorta vascular cells includes, for
example, the following method.
[0055] Human aorta vascular cells are lysed preferably using SDS or
protenase K, and DNA is deproteinized by repetitive extraction with
phenol. RNA is preferably digested with ribonuclease. The obtained
DNA is partially digested with a suitable restriction enzyme and
the obtained DNA fragment is amplified by a suitable phage or
cosmid to form a library. Then, the clone having the desired
sequence is detected by, for example, a method using a DNA probe
with a radioactive label, and a whole or partial PGIS gene is
cleaved from said clone by using a restriction enzyme and the
like.
[0056] (3) The DNA of the present invention can be prepared by
chemical synthesis by a conventional method based on the nucleotide
sequence depicted in Sequence Listing Sequence No. 11.
[0057] The present invention further relates to a recombinant
vector comprising DNA encoding the above-mentioned PGIS. The
recombinant vector of the present invention is not particularly
limited as long as it can be retained by replication or
self-proliferation in various prokaryotic and/or eukaryotic host
cells, and includes plasmid vector and phage vector.
[0058] The recombinant vector can be easily prepared by ligating
the DNA encoding human-originated PGIS of the present invention
with a commercially available recombinant vector (plasmid DNA and
bacteriophage DNA) by a conventional method. Usable recombinant
vector includes, for example, Escherichia coli-originated plasmids
pBR322, pBR325, pUC12 and pUC13; yeast-originated plasmids pSH19
and pSH15; and Bacillus subtilis-originated plasmids pUB110, pTP5
and pC194. Examples of phage include bacteriophage such as .lambda.
phage, and animal or insect viruses such as retrovirus, vaccinia
virus, nuclear polyhedrosis virus and adenovirus [e.g. pVL1392,
pBK283, Autographa californica nuclear polyhedrosis virus (AcNPV)
and Bombyx mori nuclear polyhedrosis virus (BmNPV)].
[0059] When production of PGIS by the expression of the PGIS gene
is aimed, an expression vector is useful. The expression vector is
not particularly limited as long as it expresses the PGIS gene in
various prokaryotic and/or eukaryotic host cells and is capable of
producing proteins. Preferred are that derived from insect virus
which infects insect cells and produces PGIS in said cells, and
that derived from animal virus which infects animal cells and
produces PGIS in said cells.
[0060] When bacteria, particularly Escherichia coli, is used as the
host cell, the expression vector generally consists of at least
promoter-operator region, initiation codon, DNA encoding the PGIS
of the present invention, termination codon, terminator region and
replicon.
[0061] When yeast, animal cell or-insect cell is used as the host
cell, the expression vector preferably consists of at least
promoter, initiation codon, DNA encoding the polypeptide of the
present invention and termination codon. It may contain DNA
encoding signal peptide, enhancer sequence, non-translation region
on the 5' or 3' side of the polypeptide of the present invention,
splicing junction, polyadenylation site, selection marker region,
replicon and the like.
[0062] The promoter-operator region for expressing the polypeptide
of the present invention in bacteria contains promoter, operator
and Shine-Dalgarno (SD) sequence such as AAGG. When the host is
Escherichia coli, the region preferably contains, for example, Trp
promoter, lac promoter, recA promoter, .lambda.PL promoter and lpp
promoter. The promoter for expressing PGIS in yeast includes, for
example, PH05 promoter, PGK promoter, GAP promoter and ADH
promoter, and when the host is bacteria belonging to the genus
Bacillus, SL01 promoter, SP02 promoter and penP promoter can be
used. When the host is eukaryotic cells such as animal cells,
examples of the promoter include, but not limited to, SV40-derived
promoter, retrovirus promoter, heat shock promoter, polyhedron
promoter that a nuclear polyhedrosis virus has, cytomegalovirus
promoter, adenovirus promoter and .beta.-actin promoter. The use of
an enhancer is also effective for the expression.
[0063] Preferable initiation codon includes, for example,
methionine codon (ATG).
[0064] The termination codon is exemplified by conventional
termination codons such as TAG and TGA.
[0065] As the terminator region, conventional intact or synthetic
terminator can be used.
[0066] By replicon is meant a DNA capable of reproducing the entire
DNA sequence in the host cell, and exemplified by naturally
occurring plasmid, artificially modified plasmid (DNA fragment
prepared from naturally occurring plasmid) and synthetic plasmid.
Examples of preferable plasmid include plasmid pBR322 and
artificial modification thereof-(DNA fragment obtained by treating
pBR322 with a suitable restriction enzyme) in the case of E. coli;
yeast 2 .mu. plasmid and yeast chromosomal DNA in the case of
yeast; and plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145,
plasmid pdBPV-MMTneo ATCC 37224 and plasmid pSV2neo ATCC 37149 in
the case of mammalian cell.
[0067] Enhancer sequence, polyadenylation site and splicing
junction site can be those conventionally used by artisan, such as
respective ones derived from SV40.
[0068] As the selection marker, conventional ones can be used
according to a conventional method. Examples thereof include a gene
resistant to antibiotic such as tetracycline, ampicillin and
kanamycin.
[0069] The expression vector of the present invention can be
prepared by ligating at least the above-mentioned promoter,
initiation codon, DNA encoding PGIS of the present invention,
termination codon and terminator region sequentially and cyclically
into a suitable replicatable unit. For this end, suitable DNA
fragments such as linker and other restriction sites can be used by
a conventional method such as digestion with restriction enzyme and
ligation using T4DNA ligase on demand.
[0070] The transformant of the present invention can be prepared by
introducing the above-mentioned expression vector into a host
cell.
[0071] Examples of the host cell include microorganisms such as
bacteria (e.g. bacteria belonging to the genera Escherichia and
Bacillus), yeast such as those belonging to the genus
Saccharomyces, animal cells and insect cells. Specifically
exemplified are Escherichia coli K12DH1, M103, JA221, HB101, C600,
XL-1 Blue and JM109 as the bacteria belonging to the genus
Escherichia; and Bacillus subtilis 207-21 as the bacteria belonging
to the genus Bacillus. Examples of the yeast include' Saccharomyces
cerevisiae AH22, AH22R-, NA87-11A and DKD-5D. Examples of animal
cell include simian cell COS-7, Vero, Chinese hamster cell CHO,
mouse L cell, human FL cell and human 293 cell. Examples of insect
cell include BmN4 and Sf9. Preferred are insect cells and animal
cells.
[0072] The preferred host cell for cloning the DNA sequence and
constructing the vector is generally a prokaryotic cell. The
expression vector constructed is used to transform a suitable host
cell. The host cell may be a prokaryotic cell or an eukaryotic cell
as well. Preferred are insect cells (e.g., BmN4 and Sf) and animal
cells.
[0073] The expression vector is introduced (i.e., transformation
which is used in a concept inclusive of transfection in the present
invention) into host cells by a conventionally known method.
[0074] For example, in the case of bacteria (e.g. Escherichia coli
and Bacillus subtilis), the method of Cohen et al [Proc. Natl.
Acad. Sci. USA., 69, 2110 (1972)], protoplast method [Mol. Gen.
Genet., 168, 111 (1979)] or competent method [J. Mol. Biol., 56,
209 (1971)] may be used; in the case of Saccharomyces cerevisiae,
the method of Hinnen et al [Proc. Natl. Acad. Sci. USA., 75, 1927
(1978)] or lithium method [J. Bacteriol., 153, 163 (1983)) may be
used; in the case of animal cells, the method of Graham [Virology.,
52, 456 (1973)], lipofectin method or HVJ-liposome method
[Hypertension, 21, 894-899 (1993)] may be used; and in the case of
insect cells, the method of Summers et al [Mol. Cell. Biol., 3,
2156-2165 (1983)) may be used for transformation.
[0075] The human-originated PGIS of the present invention can be
prepared by culturing, in a nutrient medium, a transformant (which
term is used in a concept inclusive of transfectant in the present
invention) comprising the expression vector prepared as in the
above.
[0076] The nutrient medium preferably contains carbon source,
inorganic nitrogen source or organic nitrogen source necessary for
the growth of host cell (transformant). Examples of carbon source
include glucose, dextran, soluble starch and sucrose; examples of
inorganic nitrogen source or organic nitrogen source include
ammonium salts, nitric acid salts, amino acid, porn steep liquor,
peptone, casein, meat extract, soybean meal and potato liquid
extract. When desired, other nutrients such as inorganic salt (e.g.
calcium chloride, sodium dihydrogenphosphate and magnesium
chloride), vitamins, and antibiotics such as ampicillin and
kanamycin may be added to the medium.
[0077] Culture is carried out according to the method known in the
pertinent field. Culture conditions such as temperature, pH of the
medium and culture time are appropriately determined so that the
maximum potency of PGIS can be obtained.
[0078] Specific media and culture conditions to be employed
according to the host cell are exemplified in the following, which
are not limitative.
[0079] When the host is bacteria, Actinomyces, yeast or filamentous
fungus, for example, liquid media containing the above-mentioned
nutrient sources are appropriate. Preferred is a medium having a pH
of 5-8.
[0080] When the host is Escherichia coli, preferable medium is M9
medium [Miller, J., Exp. Mol. Genet., p. 431, Cold Spring Harbor
Laboratory, New York (1972)]. In this case, culture is performed
with aeration and agitation as necessary, at 14-43.degree. C. for
about 3 to 24 hours.
[0081] When the host is bacteria belonging to the genus Bacillus,
culture is performed with aeration and agitation as necessary, at
30-40.degree. C. for about 16 to 96 hours.
[0082] When the host is yeast, the medium is exemplified by
Burkholder minimum medium [Bostian, K. L. et al, Proc. Natl. Acad.
Sci. USA, 77, 4505 (1980)] which preferably has a pH of 5-8.
Culture is generally performed at about 20-35.degree. C. for about
14 to 144 hours with aeration and agitation where necessary.
[0083] When the host is animal cell, the medium is exemplified by
MEM medium containing fetal calf serum at about 5-20% [Science,
122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI1640
medium [J. Am. Med. Assoc., 199, 519 (1967)] and 199 medium [Proc.
Soc. Exp. Biol. Med., 73, 1 (1950)]. The pH of the medium is
preferably about 6-8, and culture is generally performed at about
30-40.degree. C. for about 15-60 hours with aeration and agitation
where necessary.
[0084] When the host is insect cell, the medium is exemplified by
Grace's medium containing fetal calf serum [Proc. Natl. Acad. Sci.
USA, 82, 8404 (1985)] which preferably has a pH of about 5-8.
Culture is generally performed at about 20-40.degree. C. for about
15 to 100 hours with aeration and agitation where necessary.
[0085] The human-originated PGIS of the present invention can be
recovered as in the following from the culture obtained above.
[0086] That is, when the human-originated PGIS is present in the
liquid portion of the culture, the culture thus obtained is
subjected to filtration or centrifugation to separate culture
filtrate (supernatant), and PGIS is purified and separated from
said culture filtrate by a conventional method employed for
purifying and isolating natural or synthetic proteins.
[0087] The method for purification and isolation includes, for
example, a method utilizing the solubility, such as salting out and
solvent precipitation, a method utilizing the difference in
molecular weights such as dialysis, ultrafiltration, gel filtration
and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a
method utilizing charge such as ion exchange chromatography and
hydroxyapatite chromatography, a method utilizing specific affinity
such as affinity chromatography, a method utilizing the difference
in hydrophobicity such as reversed phase high performance liquid
chromatography and a method utilizing difference in isoelectric
point such as isoelectric focusing.
[0088] When the human-originated PGIS is present in the periplasm
or cytoplasm of the cultured transformant, the culture is subjected
to a conventional method such as filtration and centrifugation to
collect the cells; the cells are suspended in a suitable buffer and
subjected to lysis of cell wall and/or cell membrane by
ultrasonication, using lysozyme or by freeze-thawing; and the
membrane fraction containing PGIS is obtained by centrifugation or
filtration. Said membrane fraction is solubilized with surfactant
such as Triton to give a crude solution. The crude solution is
treated by a conventional method as exemplified supra to isolate
and purify PGIS of the present invention.
[0089] The present invention also relates to an antibody having a
reactivity with the above-said human-originated PGIS. The antibody
of the present invention encompasses both the polyclonal antibody
and monoclonal antibody having the above-mentioned properties. The
antibody of the present invention can be obtained by a conventional
method.
[0090] For example, the monoclonal antibody of the present
invention can be prepared from hybridoma produced by so-called cell
fusion. That is, fused hybridoma is formed from the
antibody-producing cell and bone marrow cell; said hybridoma is
cloned; and a clone is selected which produces an antibody having a
specific affinity for an antigen, i.e. a polypeptide having part or
whole of the human-originated PGIS amino acid sequence. The
procedure therefor may be known methods except the use of the
human-originated PGIS of the present invention as an immunizing
antigen.
[0091] The immunogen can be used for immunizing animals after
admixing with, for example, complete Freund adjuvant. Examples of
the animal include mouse, rat and rabbit. The animals are immunized
by subcutaneous, intramuscular or intraperitoneal injection of
about 5-200 .mu.g/injection. The immunization includes 1-4 times of
immunization at about every 1-2 weeks from the initial immunization
and final immunization at about 1-4 weeks thereafter. When about
3-5 days have passed since final immunization, antibody-producing
cells are separated from the immunized animal. The
antibody-producing cells are exemplified by spleen cells and lymph
node cells.
[0092] The bone marrow cells are, for example, those derived from
mouse, rat and human. Examples thereof include mouse myeloma
P3.X63.Ag8, P3.X63.Ag8-U1, P3.NS1-Ag4, SP2/O--Ag14 and X63-Ag8.
653. It is preferable that the antibody-producing cells and bone
marrow cells be derived from the same species of animals.
[0093] Cell fusion is performed by the method described in, for
example, Nature, vol. 266, p. 550 (1977) or an analogous method.
Specifically, it is performed using 30-50% polyethylene glycol
having an average molecular weight of 1,000-4,000 at 30-40.degree.
C. for about 1-3 minutes.
[0094] The cells obtained by cell fusion are subjected to screening
for a clone which produces the desired monoclonal antibody. That
is, the cells are cultured in, for example, a microplate and the
antibody titer of the culture supernatant in the well in which cell
growth was acknowledged is determined by, for example, enzyme
antibody method to obtain the well in which suitable antibody has
been produced. Cloning by, for example, limiting dilution from such
well gives clones. The monoclonal antibody of the present invention
can be obtained by culturing said hybridoma cell clone by
conventional culture method, high density culture method or
spinner-flask culture method and purification thereof by affinity
chromatography using protein A-bound carrier or anti-mouse
immunoglobulin-bound carrier.
[0095] Alternatively, the cultured hybridoma cells are
intraperitoneally injected to the mouse of the same species which
has been previously treated with pristance, and ascites obtained is
subjected to salting out with ammonium sulfate and DEAE ion
exchange chromatography to give purified IgG fraction containing
the same.
[0096] The DNA encoding the human-originated PGIS of the present
invention can be used for gene therapy.
[0097] The DNA encoding the human-originated PGIS of the present
invention or a recombinant vector comprising said DNA is introduced
into human or other animals, whereby PGIS is produced in the human
or other animals to promote production of PGI.sub.2. The promoted
PGI.sub.2 production in turn enables treatment (therapeutic
treatment or improvement of symptoms) of the diseases induced by a
low production of PGI.sub.2. Examples of the diseases induced by
the low production of PGI.sub.2 include cardiovascular diseases
such as thrombosis, myocardial infarction, arteriosclerosis and
angina pectoris. The recombinant vector may be introduced into
human or other animals in the form of cells transformed with said
recombinant vector.
[0098] The gene therapy utilizing the gene (inclusive of DNA and
recombinant vector) of the present invention permits setting an
appropriate environment in which the gene of the present invention
introduced into a human or other animal can fully show its
function. The treatment can be given by a conventional method as
long as it intends expression of desired effects of the gene of the
present invention. Such method is exemplified by virological means
utilizing retrovirus vector or adenovirus vector, physical means
for introducing gene by particle gun method or by using naked DNA,
and chemical means such as lipid method [Molecular Medicine, vol.
30, No. 12, p. 1526 (1993); Jikken Igaku, vol. 12, No. 3, p. 15, 28
and 40 (1994); Proc. Natl. Acad. Sci. USA, 92, 1137 (1995)]. A
method using an adenovirus vector which can be used for the gene
therapy of cystic fibrosis and which is known to permit efficient
introduction of gene into differentiated cells and tissues and
expression therein, and a method using a fusogenic liposome which
allows introduction of optional gene into tissue cells in vivo are
preferable for the gene treatment of the present invention.
[0099] The dose of the DNA or recombinant vector of the present
invention is subject to appropriate change according to sex, age
and body weight of patients, the kind of disease and symptoms
thereof, and administration route. For example, 100 .mu.g-10 mg of
DNA is generally administered.
[0100] The DNA and recombinant vector of the present invention are
administered by intravenous injection, transmucosal administration,
oral administration using enteric-coated agents, or topical
administration, with preference given to topical administration
using catheter and the like.
[0101] The DNA encoding human-originated PGIS and recombinant
vector comprising said DNA of the present invention are admixed
with conventional, pharmaceutically acceptable carrier, excipient,
diluent, extender, disintegrator, stabilizer, preservative, buffer,
emulsifier, flavor, coloring, sweetener, thickener, elixir,
solubilizer and other additives such as water, salt solution,
phosphate buffer, vegetable oil, ethanol, polyethylene glycol,
glycerol, gelatin, lactose, glucose, mannitol, starch, sucrose,
magnesium stearate, hydroxypropylcellulose, talc, lanolin and
petrolatum, and can be use used in the form of injection, tablet,
powder, capsule, enteric-coated agent, ointment, suspension,
emulsion, spray, inhalant, collunarium and the like.
[0102] A pharmaceutical composition comprising the DNA or
recombinant vector comprising said DNA of the present invention can
be administered to mammals such as human, mouse, rat, rabbit, pig,
cow, sheep, dog and cat.
[0103] Effects of the Invention
[0104] The present invention gives the first clarification of the
amino acid sequence of human-originated PGIS and nucleotide
sequence of DNA encoding the enzyme having said sequence. Based on
the elucidation of such amino acid sequence and nucleotide
sequence, the present invention provides a method for preparing
PGIS by genetic engineering and an expression system related
thereto.
[0105] The PGIS and DNA encoding same of the present invention are
useful as reagents for
[0106] (1) the analysis of physicochemical property and biological
property of PGIS at the molecular or genetic level,
[0107] (2) the analysis of the mechanism of regulating PGIS
production and the mechanism of regulating PGI.sub.2 production by
PGIS, and
[0108] (3) the investigation of the cause of various cardiovascular
diseases considered to be induced by the production imbalance
between PGI.sub.2 and TXA.sub.2, and analysis at the molecular or
genetic level for the development of therapeutic agent for said
diseases.
[0109] In addition, they are useful as diagnostics for determining
the in vivo tissue expression level and distribution of PGIS or
mRNA thereof.
[0110] Moreover, they can be used as therapeutic agents for various
cardiovascular diseases such as thrombosis, myocardial infarction,
arteriosclerosis and angina pectoris, which increase the production
level of PGI.sub.2 based on lesion-specific introduction, into
human and other animals, of PGIS, DNA encoding PGIS, fragment
thereof or modified product thereof.
[0111] The expression system of PGIS comprising a recombinant
vector containing DNA encoding the human-originated PGIS of the
present invention, and a host cell transformed with said vector is
useful for the production by genetic engineering, which enables
easy and efficient mass production of human-originated PGIS.
[0112] In addition, the human-originated PGIS antibody of the
present invention serves well for the purification of
human-originated PGIS and the immunohistochemical analysis of the
cause of a disease (specific staining of various tissues such as
uterus, heart, skeletal muscle, lung and prostate).
[0113] The plasmid, enzyme such as restriction enzyme, T4DNA ligase
and other substances to be used in Examples of the present
invention are commercially available and can be used according to a
conventional method. The procedures for cloning of cDNA,
determination of nucleotide sequence, transfection of host cell,
culture of transfectant, harvesting and purification of PGIS from
obtained culture, and obtainment of antibody are well known to
those skilled in the art, or can be known from literatures.
[0114] The pHPGIS36 (PBJT-BA-4, deposit number FERM BP-4653) and
pHPGIS135 (PBJT-BA 5, deposit number FERM BP-4654) used in the
present invention are at international deposit at National
Institute of Bioscience and Human-Technology Agency of Industrial
Science and Technology.
EXAMPLES AND REFERENCE EXAMPLES
[0115] The present invention is described detailedly in the
following by way of Examples and Reference Examples, to which the
present invention is not limited.
Example 1
[0116] Determination of cDNA Nucleotide Sequence
[0117] (1) Preparation of .lambda. hPGIS141
[0118] Human genomic library (Genomic lung fibroblast cell line, W
I 38, manufactured by Clone-Tech) was seeded at about
2.times.10.sup.5 PFU, and screened by plaque hybridization using,
as a probe, a bovine cDNA prepared in advance by the inventor (see
Tanabe, T., Hara, S., Miyata, A., Brugger, R., and Ullrich, V.
(1993) in Abstract book of 3rd international conference on
eicosanoid and other bioactive lipids in cancer, inflammation and
radiation Injury, pp. 137).
[0119] As a result, four positive signals were obtained, one of
which was isolated to a single plaque. Liquid culture thereof
resulted in mass preparation of phage DNA. After purification, it
was digested with various restriction enzymes, followed by mapping.
A fragment comprising exon was identified by Southern
hybridization, which was followed by structural analysis by DNA
sequencing to confirm that the finally-isolated clone (.lambda.
hPGIS141) coded for human PGIS.
[0120] The .lambda. hPGIS141 thus obtained was structurally
analyzed by restriction enzyme site mapping and nucleotide sequence
determination, and it was found that .lambda. hPGIS141 contained
the region corresponding to a 673rd-855th nucleotide sequence of
bovine PGIS cDNA (SQ No. 8).
[0121] Based on the nucleotide sequence of .lambda. hPGIS141 cDNA
fragment thus obtained, primers [SQ No. 1: P1 primer (674-689), SQ
No. 2: P2 primer (699-718), SQ No. 3: P3 primer (696-713), SQ No.
4: P4 primer (805-822)] having the sequences depicted in Sequence
List SQ Nos. 1-4 were synthesized.
[0122] (2) Amplification of cDNA by PCR Method
[0123] The 3'-downstream region and 5'-upstream region of cDNA were
amplified by PCR method (Biochem. Biophys. Res. Commun. 178,
1479-1484 (1991)) using said primers and poly(A).sup.+ RNA (mRNA)
from 1 .mu.g of human aorta vascular endothelial cells (hereinafter
referred to as HAEC, manufactured by Kurabo) as a template.
[0124] For amplification of cDNA corresponding to the 3'-downstream
region, cDNA was primed with a dT.sub.17 adapter
(5'-GACTCGAGTCGACATCGA-(- T).sub.17-3', SQ No. 5), and elongated to
give a first cDNA chain which was amplified with P1 primer
(674-689) and the adapter primer (SQ No. 6), and then with P2
primer (699-718) and the adapter primer (SQ No. 6). The 5'-upstream
region of the cDNA was amplified using a 5' RACE system (GIBCO
BRL). According to the protocol, homomeric dC tail was added to the
first cDNA chain and a second cDNA chain was formed using an
adapter primer (5'-(CUA).sub.4
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') (SQ No. 7). The fist step
amplification was performed using P4 primer and the adapter primer
(SQ No. 7). The second step amplification was performed using P3
primer and the adapter primer (SQ No. 7). The PCR method was
repeated 35 cycles according to the following cycloprofile.
1 Denaturation 94.degree. C., 1 minute Annealing 54.degree. C., 1
minute Elongation 72.degree. C., 3 minutes
[0125] The respective PCR products (3'-downstream region
amplification product and 5'-downstream region amplification
product) were partially taken out and purified by electrophoresis
using 1% agarose gel. Southern hybridization was applied using
bovine cDNA (pBPGISI) as a probe, and DNA was extracted from the
band which cross-hybridized to said probe. The obtained DNA was
cloned into pBluescriptII SK(-).
[0126] That is, cloning and screening were performed by the
following steps:
[0127] (1) cleaving out the band which showed a signal from a gel,
after electrophoresis
[0128] (2) agarase digestion at 40.degree. C. for one hour (agarase
1 unit/100 .mu.l gel)
[0129] (3) extraction of DNA with phenol and subsequent ethanol
precipitation
[0130] (4) dissolving said DNA ethanol precipitate in sterile water
and treating with polynucleotide kinase at 37.degree. C. for one
hour
[0131] (5) end repairing with Klenow fragment (16.degree. C., 1
hr)
[0132] (6) ligation using Takara ligation kit
[0133] (7) transformation by a conventional method
[0134] (8) sewing in a plate
[0135] (9) forming a replica by a conventional method and
[0136] (10) colony hybridization of nitrocellulose filter of the
replica by a conventional method, using bovine PGIS cDNA as a
probe
[0137] The hybridization was performed at 60.degree. C. in
6.times.SSC [1.times.SSC containing 0.15 M NaCl, 15 mM sodium
citrate (pH 7.0)], 5.times. Denhardt's solution, 250 .mu.g/ml
salmon sperm DNA, 0.1% SDS and cDNA fragment (10.sup.6 cpm/ml)
labeled by random priming method. The filter obtained was washed
twice with 3.times.SSC and 0.1% SDS at room temperature for 5
minutes and twice with 0.1.times.SSC and 0.1% SDS at 50.degree. C.
for 15 minutes. The filter was air-dried, and exposed to Fuji X ray
film using a intensifying screen at -80.degree. C. for 12-16
hr.
[0138] The obtained DNA insert was subcloned into pBluescriptII
SK(-). By these steps, a clone (pHPGIS135) containing 3'-downstream
region DNA of human-originated PGIS and a clone (pHPGIS36)
containing 5'-upstream region DNA of human-originated PGIS were
obtained. Then, the nucleotide sequence of the DNA insert of
respective clones was determined by the Sanger method (Sanger, F.,
Nickle,, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA
74, 5463-5467] using Taq dye primer cycle sequence kit
(manufactured by Applied Biosystems) and Model 373A DNA sequencer
(manufactured by Applied Biosystems). As a result, it was found
that pHPGIS36 clone had, as a DNA insert sequence, a 740 bp
nucleotide sequence (SQ No. 9) of cDNA of human PGIS, having an
adapter sequence on the 5' side, based on which partial amino acid
sequence of PGIS comprising 238 amino acid residues wherein ATG is
the translation initiation sequence (Met) was identified.
[0139] It was also found that pHPGIS135 clone comprised, as a DNA
insert sequence, a 1277 bp nucleotide sequence (SQ No. 10) of cDNA
of human PGIS, having an adapter sequence on the 3' side, based on
which partial amino acid sequence of PGIS on the carboxyl side
region starting from 226th aspartic acid was identified. The
nucleotide sequence of human PGIS cDNA contained in pHPGIS36 clone
and the amino acid sequence deduced therefrom are depicted in SQ
No. 9 in the Sequence Listing to be mentioned later, and the
nucleotide sequence of human PGIS cDNA contained in pHPGIS135 clone
and the amino acid sequence deduced therefrom are depicted in SQ
No. 10 therein. FIG. 1 shows a restriction enzyme map of human PGIS
cDNA and the region of human PGIS cDNA, which corresponds to the
DNA contained in .lambda. hPGIS141, pHPGIS36 and pHPGIS135. FIG. 2
shows a restriction enzyme map of pHPGIS36 and FIG. 3 shows a
restriction enzyme map of pHPGIS135.
[0140] Human PGIS cDNA obtained by the above-mentioned cloning had
a consensus sequence of the initiation codon of eukaryotic shown by
Kozak et al [Nucleic Acids Res. 12, 857-872 (1984)] at around the
translation initiation codon, and TGA codon corresponding to the
termination codon at 500 codons therefrom. Based on these facts, it
was found that the cDNA of the cloned human PGIS comprised 1977 bp
comprising 1500 bp encoding 500 amino acid residues, as shown in SQ
No. 12, and the molecular weight of the protein coded thereby was
speculated to be about 57,000.
[0141] Comparison of the amino acid sequence encoded by said DNA
with the amino acid sequence of bovine-originated PGIS separately
cloned by the present inventor revealed an about 88% homology. The
study of bovine PGIS by the present inventor found that the bovine
PGIS had a 31% homology with-cholesterol 7.alpha.-hydroxylase
belonging to the cytochrome P450 7 family (CYP7), and the region
around the 441st Cys residue, which is heme-binding site (fifth
ligand) of cytochrome P450, was reserved. The human PGIS similarly
reserved the amino acid sequence corresponding to said region, and
this region is considered to play an important role in the PGIS
activity.
[0142] Although the bovine PGIS had a 31% homology with
cholesterol, 7.alpha.-hydroxylase, it had only a 16% homology with
human thromboxane synthase belonging to the cytochrome P450 family
and a not more than 40% homology with any of the known cytochrome
P450 proteins. It is postulated, therefore, that it is a new family
in the cytochrome P450 super family, and human PGIS also belongs to
this new family.
[0143] A search for such structural correlation in activity is
indispensable for the study and development of pharmaceutical
products. Such search is accomplished only after the primary
structure of human PGIS has been clarified. Accordingly, the
present invention which discloses the primary structure of human
PGIS for the first time is extremely important and significant for
the research, and from industrial aspect as well.
Example 2
[0144] Expression of Human PGIS
[0145] (1) Construction of Expression Vector for Human PGIS
[0146] A cDNA insert region is cleaved out respectively from the
obtained pHPGIS36 clone and pHPGIS135 clone using a suitable
restriction enzyme, and purified. The both fragments obtained were
thermally denatured (95.degree. C. for 10 minutes), followed by
annealing. cDNA is replicated using a DNA polymerase to the both
directions toward 5' and 3' from the overlapped region as the
synthesis initiation region. Using the obtained whole length cDNA
as a template, a primer is synthesized from each region of
initiation codon or termination codon and PCR is performed. On this
occasion, a suitable restriction enzyme site is constructed as an
anchor site at 3' of the primer.
[0147] The PCR product thus obtained is purified, the nucleotide
sequence of which is confirmed, and the product is digested with
BamHI and SmaI (BglII) to give a BamHI-SmaI (BglII) fragment. Said
BamHI-SmaI (BglII) fragment is introduced into the BamHI-SmaI site
of pVL1393 expression vector previously treated with BamHI-SmaI.
The recombinant plasmid thus formed (PGIS7) is characterized by
restriction enzyme mapping and DNA sequence analysis.
[0148] (2) Baculovirus Expression System
[0149] Sf9 cells (manufactured by In Vitrogen) are mono-layer
cultured in a Grace's insect medium containing 10% fetal calf
serum, 0.33% yeastolate and 0.33% lactoalbumin hydrolysate at
27.degree. C. For the production-of a recombinant virus, Sf9 cell
(1.5.times.10.sup.6 cells) recombinant plasmid (PGIS7, 50 .mu.g)
and wild type baculovirus DNA (AcNPV; 1 .mu.g) are mixed and
transfected by calcium phosphate precipitation method. The
recombinant baculovirus is isolated and amplified by a combination
of plaque assay and slot hybridization using a .sup.32P-labeled
cDNA fragment of PGIS as a probe.
[0150] Said Sf9 cells are infected with wild type baculovirus or
recombinant baculovirus. At 3 days after the infection, cells are
collected (2.times.10.sup.8 cells) and incubated for 5 hours in a
serum-containing medium with or without 10 .mu.M hemin.
[0151] The obtained cells are washed with phosphate-buffered saline
and preserved at -80.degree. C. The microsomal fraction of the cell
is prepared according to the method of Haurand and Ullrich et al.
(J. Biol. Chem. 260, 15059-15067). The obtained cells
(2.times.10.sup.8 cells ) are homogenized in a solution (20 ml) of
10 mM potassium phosphate buffer (pH 7.0), 10 mM EDTA, 5 mM
glucose, 0.1 mM dithiothreitol (DTT), 1.15% KCl, 2 .mu.g/ml
leupeptin, 2 .mu.g/ml pepstatin, 10 .mu.g/ml soybean trypsin
inhibitor and 44 .mu.g/ml phenylmethylsulfonyl fluoride, and
subjected to ultrasonication (30 seconds, 4 times) using a Branson
sonifier model 450.
[0152] The obtained homogenate is centrifuged at 7,000.times.g for
15 minutes, and the obtained supernatant is centrifuged at
105,000.times.g for 60 minutes. The sediment obtained is suspended
in 10 mM potassium phosphate buffer (3 ml, pH 7.0) containing 20%
glycerol, 1 mM DTT and 1 mM EDTA by sonication. The protein
concentration is determined by Lowry method using bovine serum
albumin as a standard, and a solution for immunoblot analysis and
PGIS assay at 5 mg/ml is prepared.
[0153] (3) Western Immunoblot Analysis
[0154] The infected Sf9 cells and human platelet microsomal
fraction are subjected to 10% SDS-PAGE according to the method of
Laemmli [Nature 227, 680-685 (1979)]. The migrated protein is
electrophoretically transferred onto a polyvinylidene difluoride
(PVDF) membrane (Immobilon, Millipore) according to the method of
Towbin et al. [Proc. Natl. Acad. Sci. USA 76,4350-4354 (1979)].
Tris-HCl buffered saline (TBS) (pH 7.4) containing 10% equine serum
is pretreated at room temperature for 30 minutes, and the blot
membrane is incubated with polyclonal antibody against bovine PGIS
in TBS containing 3% skim milk.
[0155] After washing with TBS containing 0.05% Tween 20, the
membrane is incubated in TBS containing 3% skim milk at 37.degree.
C. for 30 minutes together with anti-mouse IgG equine antibody
conjugated with horseradish peroxidase (manufactured by Vector
Laboratories). After thorough washing with TBS containing 0.05%
Tween 20, the band showing positive immunological response is
detected using an immunostaining HRP kit (manufactured-by
Konica).
Example 3
[0156] Expression of Human PGIS in Cultured Animal Cell
[0157] (1) Preparation of Whole Length Human PGIS cDNA
[0158] The obtained pHPGIS36 clone was cleaved out with restriction
enzymes SalI and NspI and purified to give a SaLI-NspI fragment.
The pHPGIS135 clone was cleaved out with restriction enzymes PstI
and BamHI and purified to give a PstI-BamHI fragment. Furthermore,
primers [SQ No. 13: P5 primer (676-699), SQ No. 14: P6 primer
(832-855)] having sequences depicted in Sequence Listing Sequence
Nos. 13 and 14 were synthesized based on the nucleotide sequence of
.lambda. hPGIS141. Using these primers and .lambda. hPGIS141 as a
template, a middle stream region of human PGIS cDNA was amplified
by PCR method, cleaved with restriction enzymes NspI and PstI,
purified and confirmed for the nucleotide sequence and used as an
NspI-PstI fragment. These SalI-NspI fragment, PstI-BamHI fragment
and NspI-PstI fragment were bound and introduced into the
SalI-BamHI site of pBluescriptII SK+ (manufactured by STRATAGENE)
previously treated with SalI-BamHI, whereby a plasmid (pHPGIS1)
containing the whole length human PGIS cDNA was prepared. FIG. 4
shows the restriction enzyme map of pHPGIS 1.
[0159] (2) Construction of Human PGIS Expression-Vector for
Cultured Animal Cell
[0160] Human PGIS cDNA insert region was cleaved out from the
obtained PHPGIS1 clone with restriction enzymes SalI and BamHI and
purified to give a SalI-BamHI fragment. This SalI-BamHI fragment
was introduced into the SalI-BamHI site of pCMV7 expression vector
[supplied by Dr. David W. Russel, University of Texas Southwestern
Medical Center, Cell, 75, 187-197 (1993); J. Biol. Chem., 264,
8222-8229 (1989)] previously treated with SalI-BamHI, whereby a
human PGIS expression vector (pCMV-HPGIS 1) for cultured animal
cell was prepared. FIG. 5 shows the restriction enzyme map of
PCMV-HPGIS 1.
[0161] (3) Expression of Human PGIS in Cultured Animal Cell
[0162] Human fetus kidney-derived 293 cells (manufactured by
Dainippon Pharmaceutical Co., Ltd.) were sewn in a 60 mm dish at
3.times.10.sup.5 cells, and mono-layer cultured at 37.degree. C.
for 24 hours in Dulbecco modified Eagle's medium (DMEM) containing
10% fetal calf serum, 100 U/ml penicillin and 100 .mu.g/ml
streptomycin. Then, a recombinant plasmid (pCMV-HPGIS 1, 3 .mu.g)
and pVA1 [adenovirus VA1 gene, 3 .mu.g: supplied by Dr. David W.
Russel, University of Texas Southwestern Medical Center; Mol. Cell.
Biol., 7, 549-551 (1987)] were mixed and transfected by lipofectin
method (GIBCO BRL). At 40 hours after the transfection, the cells
were washed with phosphate-buffered saline and collected. The cells
were suspended in 10 mM calcium phosphate buffer (pH 7.0)
containing 10 Em EDTA, 10 mM phenylmethanesulfonyl fluoride (PMSF),
5 mM glucose, 0.1 mM dithiothreitol (DTT), 1.15% KCl, 2 .mu.g/ml
leupeptin, 2 .mu.g/ml pepstatin and 10 .mu.g/ml soybean trypsin
inhibitor, and subjected to ultrasonication (10 seconds, 10 times)
using ASTRASON.TM. Model XL2020.
[0163] The obtained homogenate was centrifuged at 100,000.times.g
for 60 minutes and the obtained sediment was suspended in 10 mM
calcium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM PMSF,
20% glycerol and 0.1 mM DTT. The protein concentration of the
obtained sample was determined using a BCA (bicinchoninic acid)
protein concentration determination kit (manufactured by PIERCE)
using bovine serum albumin as a standard. The PGIS activity of the
obtained sample was determined by reacting same with
.sup.14C-labeled PGH.sub.2 (5 nmole) as a substrate at 24.degree.
C. for 2 minutes, separating 6-keto-PGF.sub.1.alpha., which is a
metabolite of the produced PGI.sub.2, by thin layer chromatography,
and detecting the radioactivity of the 6-keto-PGF.sub.1.alpha..
FIG. 7 shows the detected PGIS activity, FIG. 8 shows PGIS activity
of positive control (bovine platelet microsomes) and FIG. 9 shows
the analysis results, by thin layer chromatography, of negative
control wherein pCMV7 alone was introduced.
[0164] As the result of the determination using a sample prepared
from the cell into which an expression vector incorporating human
PGIS cDNA had been introduced, a spot of 6-keto-PGF.sub.1.alpha.,
which is a metabolite of PGI.sub.2, was detected as shown by an
arrow in FIG. 7. The results were the same as those obtained using
bovine platelet microsome containing PGIS as a positive control
(FIG. 8). In contrast, the determination using a sample prepared
from the cell into which an expression vector without human PGIS
cDNA had been introduced failed to detect a spot of
6-keto-PGF.sub.1.alpha.. The spot of PGH.sub.2 was thicker (FIG. 9)
than in FIG. 7 and FIG. 8. The above results mean that PGIS cDNA
incorporated in the expression vector was expressed as a
recombinant protein (recombinant PGIS) having PGIS activity and
this protein acted on PGH.sub.2 to produce 6-keto-PGF.sub.1.alpha.
which is a metabolite of PGI.sub.2.
Example 4
[0165] Expression of Human PGIS in Cultured Animal Cell
[0166] Human PGIS cDNA was bound to the XhoI site of the expression
vector pUC-CAGGS [having an enhancer of cytomegalovirus and chiken
.beta.-actin promoter] as shown in FIG. 6 [prepared according to
the description in Gene 108, 193-200 (1991)] to construct an
expression vector. Two kinds of vectors, i.e., this vector and a
vacant vector without human PGIS cDNA, were introduced into
vascular smooth muscle cells respectively prepared from rat aorta
by HVJ-liposome method [Hypertension 21, 894-899 (1993)] and
incubated in a serum-free medium [Dulbecco modified Eagle's medium
(DMEM) containing 5.times.10.sup.-7 M insulin, 50 .mu.g/ml
transferin, 0.2 mM ascorbic acid, 100 U/ml penicillin and 100
.mu.g/ml streptomycin] in a CO.sub.2 incubator at 37.degree. C. for
2 days. Then, the medium was changed to a medium containing 1% or
5% fetal calf serum (FCS), and .sup.3H-thymidine was added 16 hours
later. At 8 hours after the addition of thymidine, the thymidine
uptake was determined by a conventional method [Cancer Immunol.
Immunother. 24, 158-164 (1987)].
[0167] The results are shown in FIG. 10, wherein control was a cell
into which a vacant vector was introduced and PGIS was a cell into
which an expression vector bound with human PGIS cDNA was
introduced.
[0168] Addition of serum to the vascular smooth muscle cell
cultured in the absence of serum led to a promoted proliferation
which increased thymidine uptake. In the vascular smooth muscle
cell into which an expression vector ligated with human PGIS cDNA
was introduced, thymidine intake, namely, proliferation, was
significantly suppressed as compared to the cell into which a
vacant vector was introduced. This result suggests the possibility
of PGIS cDNA introduction suppressing abnormal growth of smooth
muscle cells in vascular intima which is observed in
arteriosclerosis and the like.
Example 5
[0169] Preparation of Anti-PGIS Polyclonal Antibody
[0170] PGIS dissolved in 0.5 ml of phosphate-buffered saline (PBS)
and an equivalent amount of adjuvant were emulsified and
subcutaneously injected to rabbit. Thereafter, similar subcutaneous
injection was given twice every 10 days, and blood was taken from
the rabbit 10 days after the final subcutaneous injection.
Anti-PGIS/IgG was purified and obtained from rabbit anti-PGIS serum
prepared from the blood of said rabbit using protein A sepharose 4B
(Bio-Rad).
Example 6
[0171] Preparation of Anti-PGIS Monoclonal Antibody
[0172] {circle over (1)} Mouse
[0173] Male inbred line BALB/c mice (5 weeks of age) were obtained
and bred on standard pellet in an animal breeding chamber
(23+1.degree. C., 70% humidity) with optional watering.
[0174] {circle over (2)} Immunogen
[0175] Human-originated purified PGIS was used. The human PGIS was,
prepared to a concentration of 1 mg/ml with Dulbecco PBS, dispensed
into test tubes by 100 .mu.g and freeze-preserved at -80.degree. C.
until use.
[0176] {circle over (3)} Immunizing Method
[0177] Human PGIS 100 .mu.g/0.5 ml and an equivalent amount of
Freund's complete adjuvant were mixed. An emulsified antigen (20
.mu.g) was administered to five male BALB/c mice (5 weeks of age)
intraperitoneally and subcutaneously at dozen sites on the back
every 2 weeks for 2 months. After the immunization for 2 months,
antibody titer was measured, and the mice having high antibody
titer were picked and applied with additional intraperitoneal
administration of 50 .mu.g, 100 .mu.g or 200 .mu.g thereof every
other week.
[0178] After the immunization for 2 months, two different mice were
intraperitoneally administered with 100 .mu.g thereof after a blank
of one month. One week later, 100 .mu.g thereof was intravenously
injected for additional immunization.
[0179] {circle over (4)} Cell Fusion
[0180] At 3 days from the final immunization, the spleen of the
BALB/c mice was removed to prepare suspensions of spleen cells in
EMEM culture medium. The spleen cells were washed 4 times with EMEM
culture medium and counted.
[0181] For cell fusion, 2-amino-6-oxy-8 azapuraine
(8-Azaguanine)-resistan- t BALB/c mouse myeloma-derived cultured
cell line (P3-X63-Ag8.cndot.653, hereinafter abbreviated as X63
cells) was used as a parent cell line. The X63 cells were
subcultured in RPMI-1640 culture medium (20 .mu.g/ml, containing
8-Azaguanine) supplemented with 5% inactivated fetal calf serum
(FCS), and X63 cells in the logarithmic growth phase were washed 3
times with RPMI-1640 culture medium and counted.
[0182] Cell fusion is performed in RPMI-1640 culture medium
containing polyethylene glycol 4000 at a concentration of 50
(w/v)%.
[0183] That is, spleen cells and X63 cells are mixed at a ratio of
10:1 and centrifuged at 1500 rpm for 5 minutes. Supernatant is
removed, and cell pellets are thoroughly suspended and subjected to
cell fusion according to the method of Kohler and Milstein using
polyethylene glycol. Thereafter, the spleen cells are suspended in
an HAT selective medium (10% FCS-added RPMI-1640 culture medium
containing 1.times.10.sup.-4 M hypoxanthine, 4.times.10.sup.-7 M
aminopterin and 1.6.times.10.sup.-5 M thymidine) so that the spleen
cells are contained at a concentration of 3.5.times.10.sup.6
cells/ml. Then, the cell suspension is dispensed into each well of
96 well microtest plate by 100 .mu.l and cultured in a carbonic
acid gas incubator (37.degree. C., 95% humidity, 8% carbonic acid
gas). On day 1 and day 2 after the initiation of culture, HAT
medium is added by one drop to each well and by 2 drops on day 7
and day 9 after the initiation of incubation, which is followed by
further culture.
[0184] {circle over (5)} Screening
[0185] From 10 days after the initiation of culture, clone cells
emerge. For confirmation of antibody production, hybridoma culture
supernatant is subjected to an antigen-antibody reaction test.
[0186] That is, 50 .mu.l each from hybridoma culture supernatant
and human PGIS antigen liquid is placed in a U-bottomed microtiter
plate and thereto is added 50 .mu.l of 20% suspension of Sepharose
4B bound with anti-mouse immunoglobulin antibody. The mixture is
stirred at room temperature for one hour and left standing for 10
minutes. After confirmation of complete sedimentation of anti-mouse
immunoglobulin antibody-bound Sepharose 4B on the bottom of the
well, 20 .mu.l of the supernatant is taken and determined for
concentration of residual human PGIS in the supernatant by PGIS
ELISA system. When anti-human PGIS monoclonal antibody against
human PGIS is present in the hybridoma culture supernatant, human
PGIS and anti-human PGIS monoclonal antibody react and anti-mouse
immunoglobulin antibody-bound Sepharose 4B sediment is formed as an
antigen-antibody complex to decrease the concentration of residual
human PGIS in the supernatant, thus proving the presence of
anti-human PGIS monoclonal antibody.
Reference Example 1
[0187] RNA Blot Analysis
[0188] RNA blot hybridization analysis was made to examine the
influence of several kinds of cytokines on the expression of
HAEC-derived human PGIS mRNA.
[0189] The entire RNA (30 .mu.g) derived from each HAEC which was
incubated for 24 hours with several kinds of cytokines [IL-1.alpha.
(1 ng/ml), IL-1.beta. (1 ng/ml), IL-6 (2.5 ng/ml), TNF-.alpha. (5
ng/ml) and TNF-.beta. (1 ng/ml)] was denatured with formamide,
electrophoresed on 1% agar gel containing 1.5% formaldehyde, and
transferred onto a nylon membrane. A probe [pHPGIS 135 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] was labeled with
[.alpha.-.sup.32P]dCTP by random priming method [Feinberg, A. P.,
and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13].
[0190] Then, hybridization was applied according to the method
described in Biochem. Biophys. Res. Commun. 178, p 1479-1484
(1991). The membrane obtained was washed with 0.1.times.SSC (0.15 M
NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.1% SDS at
60.degree. C., air-dried and autoradiographed. The results are
shown in FIG. 11. The main band of the HAEC-derived human PGIS mRNA
was found at about 6 kb and three other minor bands were found
(3.2, 2.5 and 1.7 kb). The test results revealed that the
expression of human PGIS mRNA incubated for 24 hours with
IL-1.alpha., IL-1.beta. or IL-6 increased about 2-fold as compared
with the control without cytokine treatment. Accordingly, increase
in PGI.sub.2 production caused by cytokine is considered to be
attributable to the increased expression and production of PGIS
which was achieved by cytokine. Thus, the treatment with cytokine
is an extremely useful method for increasing PGIS expression to
increase PGIS activity, which in turn accelerates PGI.sub.2
production.
Reference Example 2
[0191] In vivo Distribution of PGIS mRNA
[0192] RNA blot analysis was made to examine the distribution of
PGIS mRNA expression in human body. Specifically, a filter was
purchased from Clone-Tech on which poly (A).sup.+ RNA of various
human tissues was electrophoresed and blotted. hPGIS135 was labeled
with .sup.32P by the aforementioned method and subjected to
Northern blot hybridization under the same conditions as above.
[0193] The results are shown in FIG. 12 and FIG. 13. The results
confirm that PGIS mRNA was abundantly expressed widely in human
tissues, particularly, in uterus, heart, skeletal muscle, lung and
prostate and at significant levels, though slightly, in small
intestine, kidney, liver and brain. These results coincide with the
conventional reports of enzymatic activity and distribution in
tissue of immunological response of PGIS, thus suggesting various
biological roles assumed by PGIS besides the action in the vascular
system. The 6 kb main, strong band and 3 weak bands as shown in
FIG. 11 were observed in all tissues mentioned above, though
relative thickness among the weak bands varied between tissues.
Such various modes of presence of transcription products suggest
possible different splicing of mRNA or the presence of an analogous
gene (isozyme) as found in prostaglandin endoperoxidase.
Sequence CWU 1
1
* * * * *